Regulatory agencies including EPA, FDA, ECHA, and EMA require thorough assessment of the health effects of chemicals present in the environment and marketplace. Interpretation of positive genotoxicity findings using the current standard in vitro testing battery is a major challenge to both industry and regulatory agencies. These tests have high sensitivity, but suffer from low specificity, leading to high rates of irrelevant positive findings (i.e., positive results in vitro that are not relevant or reproduced in vivo); this leads to unnecessary and costly follow- up as well as exclusion of potentially beneficial agents from further development. We have developed an in vitro transcriptomic biomarker-based approach that provides pathway-based mechanistic context to positive genotoxicity assay data, particularly for in vitro chromosome damage assays that suffer from a high frequency of irrelevant (false positive) results. Our transcriptomic biomarker, TGx-28.65, readily distinguishes DNA damage-inducing agents from other agents with much higher accuracy. In Phase I we have successfully integrated TGx-28.65 with nCounter technology, which is based on direct multiplexed measurement of gene expression. nCounter offers high levels of precision, linearity, reproducibility, and sensitivity (<1 copy per cell). These unique characteristics of nCounter make it an ideal technology platform for our biomarker-based genotoxicity screening assay. We now show that that our nCounter approach can be used directly with cell lysates and adapted to high-throughput screening (HTS); this addresses another critical need since the standard in vitro genotoxicity panel is typically not amenable to HTS. The objective of this application is to develop and commercialize this high-throughput genotoxicity screening system using our well-validated TGx- 28.65 toxicogenomic biomarker. We will further assess our approach with priority environmental agents using 200 chemicals from the Toxcast collection. While many aneugens are not DNA-damaging, they can also trigger chromosome aberrations and micronucleus formation, so there is a need to also assess for this property. We have already observed transcriptomic responses for a limited number of aneugens and will now assess with a larger panel of aneugens with known modes of action. The proposed approach will employ our standard human TK6 cell approach with or without metabolic activation, and will be extended to HepaRG 3D spheroid culture to more closely model in vivo exposure; primary cells, such as human hepatocytes, will also be employed using the conditionally-reprogramed-cell approach developed at Georgetown. Our proposed approach can be integrated into genetic safety hazard assessment as a follow-up to positive chromosome damage findings, as well as a stand-alone for in vitro genotoxicity assessment. Considering the high cost of animal testing and now an E.U. regulatory ban for some product applications, development of accurate and cost-effective in vitro approaches is critical. This proposal should significantly benefit safety assessment by providing highly specific genotoxicity HTS, and is poised to become a commercially successful screening service.